High purity phthalein derivatives and method for preparing same

ABSTRACT

The invention concerns high purity phthalcin derivatives enabling their use for medical applications or in the field of biotechnology, as well as their preparation method whereby a phthatic anhydride derivative is condensed with a naphthol or phenol derivative in an organic acid ester and the crystals of the resulting condensate are converted by action of a strong acid or one of its precursors in anhydrous medium.

The present invention relates to high purity phthaleins and to themethod for preparing them. It relates more particularly to high purityfluorescein.

In the present description, the term “high purity phthalein” is intendedto mean a phthalein containing at most 1% by weight, preferably at most0.5% by weight, of impurities.

Phthaleins are molecules having the following xanthene unit:

These products are useful as dyes in various industries, in particularthe textile industry, the paper industry, printing, reprography, thefood industry, the cosmetics industry and the pharmaceutical industry.Phthaleins are, currently, the subject of a considerable resurgence ofinterest in the health field, for their diagnostic use, in particular inthe context of medical imaging and in the field of the biotechnology forlabeling biological molecules (nucleic acids, proteins, lipoproteins,membrane lipids) and following the intracellular or extracellularbiochemical activity of biological molecules.

For example, fluorescein is a phthalein commonly used in ophthalmologyfor performing retinal angiography by fluorescence. The diagnosticadvantage of fluorescein angiography is currently increased by theappearance of new medicinal products for treating vascular pathologiesof the retina and of the choroid and the availability of a newgeneration of retinographs that make it possible to perform digitalfluorescence imaging that has higher performance levels and givesgreater resolution than the former systems of acquisition onphotographic emulsions.

In parallel, the quality and safety requirements of the newinternational pharmaceutical standards (ICH: International Commission ofHarmonization, ICH Q Topic Q3A 1999) have considerably increased. Thesame is true with regard to the use of phthaleins in biotechnologiesthat require reagents of increasingly high quality. In order to satisfythe current requirements, the level of purity of the phthaleins used inthe pharmaceutical field or that of biotechnology must necessarily bevery high. By way of example, in the publication “Effective differencesin the formulation of intravenous fluorescein and related side effects”,Am. J. Ophthalmol. 78, 2: 217-221, 1974, L. Yannuzzi showed acorrelation between the purity of phthaleins, in particular that offluorescein, and the tolerance of these substances when they areadministered to humans by injection.

The high purity phthaleins according to the invention have the generalformula (I):

in which R1, R2, R3, R4 and R5, which are identical to or different fromone another, are chosen from the group comprising the following radicalsor groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitro,sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms,alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkylhaving from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkylhaving from 1 to 30 carbon atoms, carboxyalkyl having from 2 to 30carbon atoms, aminoalkyl having from 1 to 30 carbon atoms, sulfoalkylhaving from 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl,aryl ester, succinimidyl ester, isothiocyanate, maleimide,iodoacetamide, haloacetamide, chlorosulfonic, purine or pyrimidinebases, monosaccharides, preferably hexoses or pentoses, oligosides andpolyosides, polypeptides, proteins and phospholipids,R1 and R5 are not hydrogen when R1 is a group —CH₂—CH₂—COOH, R2 is ahydroxyl group and R4 is a group —COOH,these phthaleins containing no more than 1% by weight, preferably nomore than 0.5% by weight, and even more preferably not more than 0.2% byweight, of residual impurities.

A phthalein that is particularly advantageous, in particular forophthalmic applications, is fluorescein having such a purity.

It is known practice to prepare the phthaleins of formula (I) by thecondensation of a phthalic anhydride derivative with a phenol derivativehaving a free ortho-position with respect to a hydroxyl group.

This condensation is carried out by heating at the melting temperatureof a mixture of the phthalic anhydride and of the phenol derivative, inthe desired proportions.

This condensation can optionally be carried out in a dilution solvent.It can also be carried out in the presence of a catalyst.

In the absence of solvent, during the heating, the reaction mediumrapidly thickens and has a tendency to harden. Within the reactionmedium, areas where the temperature is too high and areas where thetemperature is not high enough then form.

In the areas where the temperature is too high, the reactants and/or thereaction product are degraded and, in the areas where the temperature isnot high enough, the reaction is not complete. The product obtained isof mediocre quality since it contains by-products that are verydifficult to remove.

In order to improve this known method, the addition of an inert solventor the use of a catalyst has been considered.

Thus, U.S. Pat. No. 1,931,049 describes the addition to the reactionmedium of an inert solvent consisting of a benzene-based or aliphatichydrocarbon, more particularly ortho-dichlorobenzene. However, thecondensation reaction is then incomplete, and generate intermediateproducts that are subsequently difficult to remove. Therefore the methoddescribed in U.S. Pat. No. 1,931,049 does not make it possible to obtaina high purity phthalein according to the definition of the presentapplication. Moreover, higher aliphatic hydrocarbons are not misciblewith the reaction medium; consequently, they introduce no improvement interms of thermal transfer and impair the elimination of the waterformed, thereby slowing down the reaction, which takes place exclusivelyin a hydrophobic medium.

Regarding the catalysts that are used to improve the reaction yield,these are concentrated sulfuric acid, anhydrous zinc chloride and tinchloride.

The German patent DE 360691, describes the use of an aromatic sulfonicacid as a catalyst, in particular benzenesulfonic acid, alone orcombined with one of the abovementioned three catalysts.

It so happens, however, that the addition of these catalysts leads to areaction product that sets and hardens with entrapment of impurities,which can no longer be eliminated from the desired product.

In order to eliminate both the generated by-products and the impurities,isolation and purification methods have been developed, but none havemade it possible to substantially improve the quality of phthaleins.

A conventional method consists in basifying the phthalein in an aqueousmedium so as to dissolve it, and then in acidifying it so as to allow itto precipitate. These two steps are repeated successively in an attemptto eliminate the impurities. However, this method does not bring about anoticeable improvement in the purity of the product since, while theimpurities dissolve with the phthalein during the basifying step, theyprecipitate again with the phthalein during the acidification step. Inaddition, this purification method has the drawback of producing aconsiderable amount of salts which are difficult and expensive tosubsequently eliminate.

U.S. Pat. No. 1,965,842 describes the purification of phthaleins,derived from hydroxybenzene, by direct extraction of the by-productswith dichlorobenzene alone or in a mixture with other solvents. Thisdirect extraction of the crude product with the solvent in question doesnot, however, make it possible to obtain extensive elimination ofimpurities, which remain partly trapped in the phthalein crystals.

None of the purification methods described in the prior art makes itpossible to reach a level of purity that is sufficient to allow thepharmaceutical use of phthaleins.

Given the advantage of these molecules for medical diagnosis, theproduction of high purity phthaleins is a real medical need and wouldsatisfy a high demand, in particular for pharmaceutical applications inophthalmology, for diagnosis, in particular in medical imaging, or inthe field of biotechnological applications (for example, as dye to labelmolecules).

The inventors have, to their credit, at the end of extensive research,found that it is possible to obtain high purity products by thecondensation of a phthalic anhydride with a phenol or naphtholderivative in a specific solvent which consists of an organic acidester.

They have also found that the use of organic acid esters as solventsmakes it possible to carry out this condensation with an excellentyield, greater than 75%.

Specifically, these solvents have the particularity

-   -   firstly, of leading to specific crystallization of the phthalein        that derives from the condensation and of excluding, from the        generated crystals, all the impurities that remain dissolved in        the reaction medium, and    -   secondly, of allowing a complete condensation that is        predominant, to detrimental and unwanted side reactions, which        results in the total consumption of the reactants and minimizes        the formation of by-products.

The method in accordance with the invention thus makes it possible toobtain high purity phthaleins with a yield that is satisfactory from anindustrial point of view.

More particularly, the invention is related to a method for preparingphthaleins, from which the residual impurities have been removed, havinggeneral formula (I):

in which R1, R2, R3, R4 and R5, which are identical to or different fromone another, are chosen from the group comprising the following radicalsor groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitro,sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms,alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkylhaving from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkylhaving from 1 to 30 carbon atoms, carboxyalkyl having from 2 to 30carbon atoms, aminoalkyl having from 1 to 30 carbon atoms, sulfoalkylhaving from 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl,aryl ester, succinimidyl ester, isothiocyanate, maleimide,iodoacetamide, haloacetamide, chlorosulfonic, purine or pyrimidinebases, monosaccharides, preferably hexoses or pentoses, oligosides andpolyosides, polypeptides, proteins and phospholipids,R1 and R5 are not representing hydrogen when R1 is a group—CH₂—CH₂—COOH, R2 is a hydroxyl group and R4 is a group —COOH,by condensation of a phthalic anhydride derivative of formula (II) witha phenol or naphthol compound of formula (III)

in which R1, R2, R3, R4 and R5 have the same meanings as above,in a solvent consisting of an organic acid ester.

Advantageously, the starting compound (III), which is condensed with thephthalic anhydride (II), is chosen from the group comprising inparticular resorcinol, orcinol, naphthol, pyrogallol, alkylaminophenoland arylaminophenol.

When resorcinol is used as starting product, the method in accordancewith the invention makes it possible to prepare fluorescein.

Advantageously, the solvent used in the method in accordance with theinvention is an organic acid ester of formula (IV)R₆—COOR₇  (IV)in which R₆ is chosen from the group comprising the following radicalsor groups: hydrogen, alkyl having from 1 to 30 carbon atoms, cycloalkylhaving from 3 to 12 carbon atoms, haloalkyl having from 1 to 30 carbonatoms, hydroxyalkyl having from 1 to 30 carbon atoms, nitroalkyl havingfrom 1 to 30 carbon atoms, aryl, aryloxy, alkylaryl, arylalkyl,substituted arylalkyl, haloaryl, aryl ester, alkyl ester having from 2to 40 carbon atoms, and alkyloxy having from 1 to 30 carbon atoms, R₇representing one of the following groups: alkyl having from 1 to 30carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, haloalkylhaving from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30carbon atoms, nitroalkyl having from 1 to 30 carbon atoms, aryl,aryloxy, alkylaryl, arylalkyl, substituted arylalkyl, haloaryl, arylester, alkyl ester having from 2 to 40 carbon atoms, or alkyloxy havingfrom 1 to 30 carbon atoms.

Particularly advantageously, the abovementioned solvent is chosen fromthe group comprising methyl, ethyl, propyl or butyl benzoates, methyl,ethyl, propyl or butyl heptanoates, methyl, ethyl, propyl or butyloctanoates, methyl, ethyl, propyl or butyl laurates, methyl, ethyl,propyl or butyl myristates and methyl, ethyl, propyl or butylpalmitates, and mixtures thereof.

The solvent is chosen according to its boiling point, so as to make itpossible to carry out the condensation reaction at a temperature ofbetween 150° C. and 250° C.

The condensation reaction can be carried out at atmospheric pressure orunder a pressure that is adjusted according to the difference thatexists between the temperature corresponding to the boiling point of thesolvent and the temperature necessary to carry out the reaction, inparticular under a pressure greater than atmospheric pressure when theboiling point of the compound (IV) is lower than the reactiontemperature.

The condensation reaction can be carried out in the presence of acatalyst chosen from the group comprising in particular Lewis acids,such as ZnCl₂ or AlCl₃, Brönsted acids such as H₂SO₄, polyphosphoricacid, or salts thereof.

Advantageously, the catalyst used is an alkali metal salt of hydrogensulfate. Particularly advantageously, the catalyst used is potassiumhydrogen sulfate (KHSO₄) or sodium hydrogen sulfate. The use of hydrogensulfate as catalyst makes it possible to obtain an excellent yield forthe condensation reaction and has the advantage, unlike other catalysts,of making it possible to obtain complete condensation of the reactants,of being able to be completely eliminated from the phthalein obtained,and of not inducing the formation of tars in the reaction medium.

At the end of the condensation reaction, a crude product is obtained,the organic purity of which is already much higher than that of theproducts obtained with the methods of the prior art, since it is equalto or greater than 95%.

However, this purity is not yet sufficient to allow pharmaceutical use,in particular by injection. Furthermore, as shown above, thepurification methods described in the prior art do not result in aprocess that makes it possible to notably improve this purity.

At the end of extensive research, surprisingly and unexpectedly, theinventors have found a method that considerably increases the purity ofcrude phthaleins resulting from the condensation reaction, by treatingthem with a strong acid or one of its precursors in an anhydrous organicmedium.

According to an advantageous embodiment, the method in accordance withthe invention consequently comprises, after the condensation reaction, astep consisting in acidifying the product resulting from thecondensation, in an anhydrous organic medium, by addition of a strongacid or one of its precursors, chosen from the group comprising inparticular sulfuric acid, hydrochloric acid, hydrobromic acid,hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate(P₂O₅), and mixtures thereof. This acidification is carried out untilthe phthalein crystals resulting from the condensation are converted tophthalein crystals having a different structure.

The effect of this acidification step is to convert the crude phthaleinthat is insoluble and dispersed in the organic medium into a form thathas a different crystalline structure and is slightly soluble in thissame medium. This conversion is very rapid, and takes place via anintermediate solubilization phase during which the impurities arereleased and completely eliminated from the phthalein crystals. This newpurification method is very advantageous since it requires very littlesolvent and yields to a very high purity in a very short time.

According to the method of the present invention, this purification stepis carried out by dispersing the crude phthalein resulting from thecondensation, in an anhydrous solvent, preferably in an alcohol, aketone, an ether or a halogenated solvent, that is either used alone oras a mixture, even more preferably in absolute ethanol or acetone, aloneor as a mixture.

The dispersion of crude phthalein thus obtained is acidified by theaddition of a strong acid or one of its precursors, chosen from thegroup comprising in particular sulfuric acid, hydrochloric acid,hydrobromic acid, hydrofluoric acid, hydriodic acid, polyphosphoricacid, pyrophosphate (P₂O₅), and mixtures thereof.

According to a particularly advantageous embodiment, the acidificationis carried out either by sparging gaseous hydrochloric acid into thephthalein dispersion, or by adding hydrochloric acid dissolvedbeforehand in an organic solvent.

According to another advantageous embodiment of the method in accordancewith the invention, the product obtained after acidification is washedwith a washing solution chosen from the group comprising water, polarsolvents such as alcohols or ketones, or slightly polar solvents such asethers and halogenated solvents, used pure or as a mixture.

At the end of the purification step, the phthaleins thus treated have apurity of greater than 98%, preferably greater than 99%, more preferablygreater than 99.5%, and even more preferably of 99.8%.

The phthaleins thus obtained have the suitable quality to prepare otherphthaleins of formula (I) by chemical modification of the groups R1, R2,R3, R4 and R5 according to the conventional methods of the art, inparticular to obtain phthaleins that can be used for labeling biologicalmolecules (nucleic acids, proteins, lipoproteins, membrane lipids) andin the field of biotechnological applications (for example, for thelabeling of molecules and of their intracellular or extracellularbiochemical activity).

The invention is particularly suitable for preparing very high purityfluorescein, i.e. such that its content of each of the by-products ofthe reaction is less than or equal to 0.2%, and preferably less than orequal to 0.1%, the sum of the contents of each of these by-productsbeing less than or equal to 0.5%.

The method for preparing a fluorescein having the abovementioned puritycomprises the following successive steps:

-   -   condensing phthalic anhydride with resorcinol, in a solvent        which is an ester of an aliphatic or aromatic organic acid,        preferably methyl benzoate, in the presence of a catalyst chosen        from the group of Brönsted acids,    -   suspending the red-colored crystals obtained in the preceding        step in an anhydrous solvent chosen from the group comprising        alcohols such as absolute ethanol, ketones such as acetone,        ethers, halogenated solvents, or mixtures thereof,    -   acidifying the suspension obtained, by the addition of a strong        acid or one of its precursors, chosen from the group comprising        sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric        acid, hydriodic acid, polyphosphoric acid, pyrophosphate (P₂O₅),        or mixtures thereof, until yellow-colored crystals are obtained,    -   washing the crystals thus obtained with a washing solution        chosen from the group comprising water, alcohols, ketones,        ethers and halogenated solvents, pure or as a mixture, until        red-colored crystals are obtained.

An X-ray diffraction analysis of the red-colored fluorescein crystals isrepresented in FIG. 1; an X-ray diffraction analysis of theyellow-colored fluorescein crystals is represented in FIG. 2; theseX-ray-diffraction analysis being obtained on the following equipment:Philips 1729 generator, Philips 1050 goniometer, Cu Kα radiation, Gonioacquisition software and Rayon processing software, under the followingoperating conditions: Voltage 40 mV Intensity 40 mA Number of points4000 Number of passages 10 Acquisition period 250 ms Start angle (°θ)3.000 End angle (°θ) 23.000 Standard silicon

According to an advantageous embodiment of the method for preparingfluorescein, the catalyst used for the condensation reaction is thehydrogen sulfate of an alkali metal, preferably potassium hydrogensulfate or sodium hydrogen sulfate.

According to another advantageous embodiment of the method for preparingfluorescein, the acidification is carried out by sparging gaseoushydrochloric acid into the fluorescein suspension or by the action, onthis fluorescein, of hydrochloric acid in solution in an anhydrousorganic solvent, preferably chosen from the group comprising alcohols,ketones, ethers and halogenated solvents, used alone or as a mixture,even more preferably isopropanol, absolute ethanol or acetone, pure oras a mixture.

Advantageously, the washing step is carried out with a mixture of water,of ethanol and of acetone.

By means of this method in accordance with the invention, it is possibleto prepare fluorescein having a purity of greater than or equal to99.7%, which provides unquestionable advantages, in particular forpharmaceutical uses in diagnosis, especially in medical imaging or elsein the field of biotechnological applications.

The inventors have demonstrated a novel crystallographic form offluorescein being yellow-colored crystals. This novel crystallographicform is identified by the X-ray diffraction analysis given in FIG. 2,which was determined under the conditions mentioned above.

They have also demonstrated a novel crystallographic form of thefollowing compounds: 4′,5′-dihydroxyfluorescein and4′,5′-dimethylfluorescein.

Thus, the invention relates to the yellow-colored fluorescein crystalsfor which the X-ray diffraction analysis is given in FIG. 2.

The invention also relates to the yellow-colored4′,5′-dihydroxyfluorescein crystals for which the X-ray diffractionanalysis is given in FIG. 4.

It also relates to the dark red-colored 4′,5′-dimethylfluoresceincrystals for which the X-ray diffraction analysis is given in FIG. 6.

All these spectra were determined with the equipment and under theconditions mentioned above.

The invention will be described in more detail with the aid of thefollowing examples which are not limiting but relate to advantageousembodiments.

EXAMPLES Example 1 Preparation of High Purity Fluorescein Synthesis ofFluorescein

A mixture comprising 55 g of resorcinol, 30 g of phthalic anhydride, 2 gof potassium hydrogen sulfate and 500 ml of methyl benzoate is broughtto 200° C. for 6 hours. After cooling, the red crystals of crudefluorescein are washed with acetone and dried.

Mass obtained=51.8 g (78%).

These crystals are analyzed by X-ray crystallography under the followingoperating conditions:

Equipment

Philips 1729 generator

Philips 1050 goniometer

Cu Kα radiation

Gonio acquisition software

Rayon processing software Conditions Voltage 40 mV Intensity 40 mANumber of points 4000 Number of passages 10 Acquisition period 250 msStart angle (°θ) 3.000 End angle (°θ) 23.000 Standard silicon

The radiocrystallogram of FIG. 1 is obtained, for which the peaks areidentified below: Theta % Number (degrees) Distance (Å) Intensities ofcounts 5.3600 8.2457 18.09 647 5.9500 7.4306 31.10 1112 6.7000 6.602036.30 1298 8.3750 5.2883 45.95 1643 9.1050 4.8675 10.79 386 9.43004.7012 14.09 504 9.9950 4.4379 8.03 287 10.9150 4.0678 9.73 348 11.62003.8241 16.41 587 11.7300 3.7888 5.40 193 12.4200 3.5813 2.82 101 13.21503.3694 100.00 3576 13.5050 3.2983 7.47 267 13.8850 3.2097 79.03 282614.9650 2.9828 5.62 201 15.3100 2.9172 3.91 140 15.7600 2.8359 11.69 41816.0000 2.7945 8.67 310 16.9100 2.6481 4.28 153 17.4750 2.5650 3.97 14217.8550 2.5122 3.80 136 18.2300 2.4622 2.52 90 18.5150 2.4256 2.96 10618.8250 2.3871 5.03 180 19.0800 2.3563 3.27 117 20.4300 2.2066 3.05 10920.6800 2.1811 4.53 162 20.9300 2.1562 3.08 110 21.1800 2.1319 2.38 8522.9400 1.9762 4.53 162

Wavelength: 1.54051 Å

Purification of Crude Fluorescein:

50 g of crude fluorescein obtained in the previous step are stirred into1000 ml of ethanol/acetone mixture. Concentrated sulfuric acid is addedto this mixture, until complete conversion of the red crystals to yellowcrystals is obtained.

The yellow crystals obtained are analyzed by X-ray crystallography asabove. The X-ray diffraction analysis of FIG. 2 is obtained, for whichthe peaks are identified below: Theta Distances % Number of (degrees)(Å) Intensities counts 3.3500 13.1813 8.55 56 4.0050 11.0283 47.18 3094.8550 9.1010 12.98 85 5.2650 8.3940 9.01 59 5.4500 8.1099 11.45 755.7900 7.6352 52.98 347 6.3000 7.0193 16.95 111 6.7700 6.5340 17.86 1177.4900 5.9090 17.56 115 8.0300 5.5140 69.77 457 8.4250 5.2572 33.13 2178.7800 5.0462 8.40 55 9.1550 4.8411 15.88 104 9.3400 4.7461 42.44 2789.6550 4.5926 34.66 227 9.8800 4.4891 24.43 160 10.1950 4.3518 57.71 37810.5800 4.1951 58.02 380 10.7350 4.1352 38.17 250 11.1650 3.9779 28.85189 11.4800 3.8701 30.23 198 11.7200 3.7919 59.85 392 12.1100 3.671626.41 173 12.3950 3.5884 58.47 383 12.6400 3.5200 100.00 655 12.94503.4384 30.84 202 13.1550 3.3845 56.64 371 13.8350 3.2211 14.66 9614.0550 3.1717 24.12 158 14.3550 3.1068 36.34 238 14.6700 3.0415 42.90281 15.1300 2.9511 42.90 281 15.7500 2.8377 18.47 121 15.9400 2.804718.17 119 16.2500 2.7526 15.42 101 16.7550 2.6719 17.71 116 16.85002.6573 19.39 127 17.2250 2.6011 18.17 119 17.7300 2.5293 15.73 10318.4200 2.4377 10.23 67 18.5750 2.4180 10.23 67 19.2650 2.3345 15.88 10419.5800 2.2984 10.84 71 19.8000 2.2739 11.91 78 20.1150 2.2397 13.28 8720.6500 2.1841 14.81 97 21.1500 2.1348 11.30 74 21.2750 2.1228 10.08 6621.8100 2.0732 7.94 52 22.2500 2.0342 9.16 60 22.6250 2.0022 9.62 6322.9100 1.9786 9.01 59

Wavelength: 1.54051 Å

These crystals are filtered off and then washed with anethanol/acetone/water (40/40/20) mixture. The washing turns the yellowfluorescein crystals to red fluorescein crystals, which have a higherpurity.

Purity by HPLC: 99.8%

Example 2 Preparation of high purity 4′,5′-dimethylfluorescein Synthesisof 4′,5′-dimethylfluorescein

A mixture comprising 62 g of 2-methylresorcinol, 30 g of phthalicanhydride, 2 g of potassium hydrogen sulfate and 500 ml of ethyl laurateis brought to 200° C. for 3 hours. After cooling, the crude product isfiltered and washed with acetone and then dried. The product obtained isa dark orange powder.

Mass obtained=49.7 g (69%)

These crystals are analyzed by X-ray crystallography in the same way asin example 1. The X-ray diffraction analysis of FIG. 3 is obtained, forwhich the peaks are identified below. Theta Distances % Number of(degrees) (Å) Intensities counts 3.5700 12.3700 7.38 81 5.0290 8.793845.49 499 6.6650 6.6365 62.26 683 6.7400 6.5630 60.07 659 7.1300 6.205787.24 957 7.4550 5.9366 20.97 230 8.3700 5.2915 6.56 72 8.9300 4.962130.63 336 9.0950 4.8728 10.12 111 9.3300 4.7511 21.24 233 9.4550 4.688919.05 209 10.0350 4.4204 12.12 133 10.3250 4.2975 18.87 207 10.60004.1873 11.49 126 10.8300 4.0994 39.65 435 11.2900 3.9344 12.49 13711.4500 3.8801 10.85 119 11.7550 3.7808 28.17 309 11.9050 3.7339 52.42575 12.2550 3.6288 79.95 877 12.7050 3.5023 8.11 89 13.2400 3.3631 21.15232 13.4200 3.3188 100.00 1097 13.7100 3.2499 10.85 119 14.5250 3.07128.84 97 14.8350 3.0084 23.25 255 15.0550 2.9654 17.59 193 15.1550 2.946315.13 166 15.3650 2.9070 25.80 283 15.7050 2.8456 20.51 225 16.19002.7625 14.13 155 16.7550 2.6719 9.02 99 17.5400 2.5558 7.47 82 17.88502.5081 8.75 96 18.1050 2.4786 6.75 74 18.4900 2.4288 19.51 214 18.79502.3907 6.11 67 18.8900 2.3791 6.84 75 19.6800 2.2872 15.50 170 20.21002.2296 7.38 81 20.3950 2.2103 9.66 106 20.6800 2.1811 6.84 75 20.99502.1498 5.38 59 21.4650 2.1049 7.11 78 21.6500 2.0878 8.84 97 21.93502.0620 9.21 101 22.1250 2.0451 9.02 99 22.5300 2.0102 5.47 60 22.78501.9889 5.93 65

Wavelength: 1.54051 Å

Purification of 4′,5′-dimethylfluorescein

40 g of crude 4′,5′-dimethylfluorescein are added to 800 ml ofethanol/acetone mixture. Concentrated sulfuric acid is added to thismixture, until complete conversion of dark orange-colored crystals toyellow-colored crystals is obtained.

The yellow crystals obtained are analyzed by X-ray crystallography asabove. The X-ray diffraction analysis of FIG. 4 is obtained, for whichthe peaks are identified below. Theta Distances % Number of (degrees)(Å) Intensities counts 3.7050 11.9199 22.22 172 4.6150 9.5732 45.61 3534.9300 8.9629 46.77 362 6.3300 6.9861 8.53 66 6.6150 6.6864 6.20 487.3000 6.0619 19.51 151 7.4500 5.9405 32.56 252 7.6500 5.7861 26.61 2068.1250 5.4499 64.21 497 8.6300 5.1332 22.48 174 9.5950 4.6211 6.59 5110.4450 4.2487 41.34 320 10.8500 4.0919 10.21 79 11.3550 3.9122 14.60113 11.7000 3.7983 9.17 71 11.7950 3.7682 9.30 72 12.2250 3.6375 23.13179 12.4500 3.5728 58.79 455 12.7650 3.4861 19.90 154 12.8950 3.451518.60 144 13.1000 3.3984 100.00 774 13.6350 3.2675 67.44 522 14.14003.1530 30.75 238 14.5250 3.0712 14.08 109 14.8400 3.0074 22.74 17615.1750 2.9425 19.38 150 15.3500 2.9098 30.23 234 15.6950 2.8474 20.93162 16.0950 2.7784 7.24 56 16.2850 2.7468 7.49 58 16.5950 2.6969 8.01 6216.9100 2.6481 14.08 109 17.3200 2.5873 8.66 67 17.5400 2.5558 7.88 6117.9150 2.5040 13.31 103 18.3550 2.4460 9.69 75 18.4800 2.4300 10.59 8218.8600 2.3828 15.37 119 19.2350 2.3380 9.43 73 19.4850 2.3092 7.75 6020.2100 2.2296 14.99 116 20.7100 2.1781 7.11 55 20.8350 2.1656 6.59 5121.0850 2.1411 6.33 49 21.5250 2.0993 12.92 100 21.6500 2.0878 8.79 6821.8400 2.0705 6.98 54 22.0900 2.0482 10.34 80 226550 1.9997 7.11 55

Wavelength: 1.54051 Å

After filtration and recrystallization from an acetone/water mixture orwashing in ethanol/acetone/water, the yellow-colored crystals turn todark orange-colored crystals.

Example 3 Preparation of 4′,5′-dihydroxyfluorescein Synthesis of4′,5′-dihydroxyfluorescein

A mixture comprising 63 g of pyrogallol, 30 g of phthalic anhydride, 2 gof potassium hydrogen sulfate and 500 ml of ethyl myristate is broughtto 200° C. for 3 hours. After cooling, the crude product is filtered andwashed with acetone and then dried. The product obtained is agrayish-brown- or anthracite-colored powder.

Mass obtained=43.5 g (59.7%)

The crystals obtained are analyzed by X-ray crystallography as above.The X-ray diffraction analysis of FIG. 5 is obtained, the peaks of whichare identified below. Theta Distances % Number (degrees) (Å) Intensitiesof counts 3.6450 12.1158 73.08 980 5.9200 7.4681 8.13 109 6.7200 6.582413.35 179 7.3350 6.0331 10.29 138 8.5050 5.2081 11.78 158 9.2300 4.802115.59 209 9.7400 4.5529 18.20 244 10.3400 4.2914 21.40 287 10.91504.0678 6.71 90 11.2600 3.9447 8.80 118 12.2550 3.6288 18.64 250 12.55503.5434 12.75 171 13.2000 3.3731 11.86 159 13.7200 3.2476 100.00 134114.7150 3.0324 17.75 238 14.8600 3.0034 13.57 182 16.1600 2.7675 6.79 9117.2550 2.5967 7.83 105 18.3550 2.4460 6.86 92

Wavelenth: 1.54051 Å

Purification of 4′,5′-dihydroxyfluorescein

40 g of crude 4′,5′-dihydroxyfluorescein are added to 800 ml ofethanol/acetone mixture. Concentrated sulfuric acid is added to thismixture, until complete conversion of grayish-brown- oranthracite-colored crystals to reddish-brown- or mahogany-coloredcrystals is obtained.

The crystals obtained are analyzed by X-ray crystallography as above.The X-ray diffraction analysis of FIG. 6 is obtained, for which thepeaks are identified below: Theta Distances % Number of (degrees) (Å)Intensities counts 3.2500 13.5865 90.53 526 3.5350 12.4923 18.76 1093.7850 11.6683 11.70 68 3.9450 11.1958 13.25 77 4.1650 10.6053 15.15 884.5400 9.7310 10.33 60 4.6350 9.5319 10.15 59 6.3950 6.9154 9.29 546.5500 6.7525 11.53 67 6.8200 6.4863 27.71 161 7.1150 6.2187 10.50 617.4800 5.9168 21.51 125 7.8700 5.6253 12.91 75 8.2200 5.3874 27.88 1628.7800 5.0462 41.48 241 9.2200 4.8073 10.15 59 9.5350 4.6499 11.70 689.7950 4.5276 42.17 245 10.1000 4.3923 17.21 100 10.5400 4.2108 16.70 9710.8850 4.0789 16.52 96 11.6500 3.8144 25.13 146 11.8900 3.7385 13.60 7912.0450 3.6911 13.08 76 12.3900 3.5898 16.52 96 12.5800 3.5365 14.80 8612.8750 3.4568 27.37 159 12.9950 3.4254 25.82 150 13.2900 3.3507 67.64393 13.5750 3.2816 27.02 157 13.7150 3.2488 27.02 157 14.1100 3.1596100.00 581 14.6200 3.0516 15.66 91 14.7750 3.0203 18.24 106 15.04002.9683 25.13 146 15.3050 2.9181 24.78 144 15.6850 2.8491 16.35 9515.9400 2.8047 11.02 64 16.2500 2.7526 10.67 62 16.5950 2.6969 12.22 7117.1650 2.6099 9.81 57 17.5700 2.5516 8.78 51 17.7600 2.5252 9.81 5718.0750 2.4826 11.36 66 18.5150 2.4256 8.43 49 18.7650 2.3944 13.08 7619.0450 2.3605 11.36 66 20.1950 2.2312 50.09 291 20.6150 2.1877 9.12 5321.0550 2.1440 12.56 73 21.4650 2.1049 9.29 54 21.6850 2.0846 9.98 5822.0600 2.0509 9.81 57

Wavelength: 1.54051 Å

After filtration and washing in water, the reddish-brown- ormahogany-colored crystals turn to grayish-brown- or anthracite-coloredcrystals.

1-22. (canceled)
 23. A phthalein of general formula (I):

wherein R1, R2, R3, R4 and R5, which are identical to or different fromone another, are selected from the group consisting of hydrogen,hydroxyl, halogen, acetyl, amino, phosphate, nitro, sulfonate, carboxyl,alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxyhaving from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbonatoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester havingfrom 2 to 40 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms,carboxyalkyl having from 2 to 30 carbon atoms, aminoalkyl having from 1to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl,aryloxy, arylalkyl, haloaryl, aryl ester, succinimidyl ester,isothiocyanate, maleimide, iodoacetamide, haloacetamide, chlorosulfonic,purine or pyrimidine bases, monosaccharides, preferably hexoses orpentoses, oligosides and polyosides, polypeptides, proteins andphospholipids, R3 and R5 are not hydrogen when R1 is a group—CH₂—CH₂-COOH, R2 is a hydroxyl group and R4 is a group —COOH, thesephthaleins containing no more than 1% by weight of residual impurities.24. The phthalein as claimed in claim 23 containing no more than 0.5% byweight of residual impurities.
 25. The phthalein as claimed in claim 24containing no more than 0.2% by weight of residual impurities.
 26. Thephthalein as claimed in claim 23 consisting of fluorescein.
 27. A methodfor preparing phthaleins, wherein the residual impurities have beenremoved, having the general formula (I):

wherein R1, R2, R3, R4 and R5, which are identical to or different fromone another, are selected from the group consisting of hydrogen,hydroxyl, halogen, acetyl, amino, phosphate, nitro, sulfonate, carboxyl,alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxyhaving from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbonatoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester havingfrom 2 to 40 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms,carboxyalkyl having from 2 to 30 carbon atoms, aminoalkyl having from 1to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl,aryloxy, arylalkyl, haloaryl, aryl ester, succinimidyl ester,isothiocyanate, maleimide, iodoacetamide, haloacetamide, chlorosulfonic,purine or pyrimidine bases, monosaccharides, preferably hexoses orpentoses, oligosides and polyosides, polypeptides, proteins andphospholipids, R3 and R5 are not hydrogen when R1 is a group—CH₂—CH₂—COOH, R2 is a hydroxyl group and R4 is a group —COOH, wherein aphthalic anhydride derivative of formula (II) is condensed with a phenolor naphthol compound of formula (III)

in which R1, R2, R3, R4 and R5 have the same meanings as above, thecondensation being carried out in a solvent consisting of an organicacid ester.
 28. The method as claimed in claim 27, wherein the compoundof formula (III) is selected from the group consisting of resorcinol,orcinol, naphthol, pyrogallol, alkylaminophenol and arylaminophenol. 29.The method as claimed in claim 27, wherein the solvent is an organicacid ester of formula (IV)R₆—COOR₇  (IV) wherein R₆ is selected from the group consisting ofhydrogen, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from3 to 12 carbon atoms, haloalkyl having from 1 to 30 carbon atoms,hydroxyalkyl having from 1 to 30 carbon atoms, nitroalkyl having from 1to 30 carbon atoms, aryl, aryloxy, alkylaryl, arylalkyl, substitutedarylalkyl, haloaryl, aryl ester, alkyl ester having from 2 to 40 carbonatoms, and alkyloxy having from 1 to 30 carbon atoms, R₇ being selectedfrom the group consisting of alkyl having from 1 to 30 carbon atoms,cycloalkyl having from 3 to 12 carbon atoms, haloalkyl having from 1 to30 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms,nitroalkyl having from 1 to 30 carbon atoms, aryl, aryloxy, alkylaryl,arylalkyl, substituted arylalkyl, haloaryl, aryl ester, alkyl esterhaving from 2 to 40 carbon atoms, or alkyloxy having from 1 to 30 carbonatoms.
 30. The method as claimed in claim 27, wherein the organic acidester is selected from the group consisting of methyl, ethyl, propyl orbutyl benzoate, methyl, ethyl, propyl or butyl heptanoate, methyl,ethyl, propyl or butyl octanoate, methyl, ethyl, propyl or butyllaurate, methyl, ethyl, propyl or butyl myristate or methyl, ethyl,propyl or butyl palmitate, and mixtures thereof.
 31. The method asclaimed in claim 27, wherein the condensation reaction is carried out atbetween 150° C. and 250° C., optionally under pressure.
 32. The methodas claimed in claim 27, wherein the reaction is carried out in thepresence of a catalyst selected from the group consisting of Lewisacids, such as ZnCl₂ or AlCl₃, Brönsted acids such as H₂SO₄ orpolyphosphoric acid.
 33. The method as claimed in claim 32, wherein thecatalyst is an alkali metal hydrogen sulfate.
 34. The method as claimedin claim 33, wherein the catalyst is potassium hydrogen sulfate (KHSO₄)or sodium hydrogen sulfate (NaHSO₄).
 35. A method for acidifying theproduct resulting from the condensation of a phthalic anhydridederivative of formula (II) with a phenol or naphthol compound of formula(III), the formulae (II) and (III) being those of claim 27, wherein thereaction is carried out in an anhydrous organic medium, by the additionof a strong acid or one of its precursors, selected from the groupconsisting of sulfuric acid, hydrochloric acid, hydrobromic acid,hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate(P₂O₅), and mixtures thereof, the acidification being carried out untilthe phthalein crystals resulting from the condensation are converted tophthalein crystals having a different structure.
 36. The method asclaimed in claim 35, wherein the condensation product is the productobtained by the method as claimed in claim
 27. 37. The method as claimedin claim 35, comprising a step consisting in washing the productobtained after acidification, said washing step being carried out with awashing solution selected from the group consisting of water, alcohols,ketones, ethers and halogenated solvents, pure or as a mixture, untilthe crystals are reconverted to the structure that they had before theacidification reaction.
 38. A method for preparing a fluorescein havinga purity such that its content of each of the by-products of thereaction is less than or equal to 0.2%, the sum of the contents of eachof these by-products being less than or equal to 0.5%, said methodcomprising the following successive steps: condensing phthalic anhydridewith resorcinol, in a solvent consisting of an ester of an aliphatic oraromatic organic acid, in the presence of a catalyst selected from thegroup consisting of Lewis acids or Brönsted acids, suspending thered-colored crystals obtained in the preceding step in an anhydroussolvent selected from the group consisting of alcohols such as absoluteethanol, ketones such as acetone, ethers, halogenated solvents, ormixtures thereof, acidifying the suspension thus obtained by theaddition of a strong acid or one of its precursors, selected from thegroup consisting of sulfuric acid, hydrochloric acid, hydrobromic acid,hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate(P₂O₅), and mixtures thereof, until the red-colored crystals areconverted to yellow-colored crystals exhibiting the X-ray diffractionanalysis of FIG. 2, washing the crystals obtained with a washingsolution selected from the group consisting of water, alcohols, ketones,ethers and halogenated solvents, pure or as a mixture, this washingbeing continued until the yellow-colored crystals are reconverted tored-colored crystals.
 39. The method for preparing a fluorescein asclaimed in claim 38, having a purity such that its content of each ofthe by-products of the reaction is less than or equal to 0.1%.
 40. Themethod for preparing a fluorescein as claimed in claim 38, wherein thesolvent used in the condensation reaction is the ethyl or methylbenzoate or ethyl or methyl palmitate.
 41. The method for preparing afluorescein as claimed in claim 37, wherein the catalyst used for thecondensation reaction is an alkali metal hydrogen sulfate.
 42. Themethod for preparing a fluorescein as claimed in claim 40, the catalystis potassium hydrogen sulfate or sodium hydrogen sulfate.
 43. The methodas claimed in claim 37, wherein the acidification is carried out bysparging gaseous hydrochloric acid into the phthalein suspension or bythe action, on this phthalein, of hydrochloric acid in solution in theanhydrous organic solvent, preferably an alcohol, a ketone, an ether ora halogenated solvent, used alone or as a mixture, even more preferablyisopropanol, absolute ethanol or acetone, pure or as a mixture.
 44. Ayellow-colored fluorescein crystal having the X-ray diffraction analysisof FIG.
 2. 45. A yellow-colored 4′,5′-dimethylfluorescein crystal havingthe X-ray diffraction analysis of FIG.
 4. 46. A reddish-brown- ormahogany-colored 4′,5′-dihydroxyfluorescein crystal having the X-raydiffraction analysis of FIG.
 6. 47. A phthalein obtained by means of themethod as claimed in claim
 27. 48. A fluorescein obtained by means of amethod as claimed in claim
 27. 49. A 4′,5′-dimethylfluorescein obtainedby means of a method as claimed in claim
 27. 50. A4′,5′-dihydroxyfluorescein obtained by means of a method as claimed inclaim
 27. 51. Pharmaceutical composition for diagnosis, especially formedical imaging comprising the fluorescein as claimed in claim
 26. 52.Pharmaceutical composition for diagnosis, especially for medical imagingcomprising the fluorescein obtained according to the method of claim 27.53. Labeling composition for biotechnological applications comprisingthe fluorescein as claimed in claim
 26. 54. Labeling composition forbiotechnological applications comprising the fluorescein obtainedaccording to the method of claim 27.